This invention relates to the field of plant genetic engineering. In particular, this invention provides DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins in transformed plastids.
Transgenic plants are useful to study nuclear gene function and regulation and to improve agronomically important crop plants. Routine application of transgenic technology is made feasible by the alternative methods developed for transformation of nuclear genomes of higher plants. However, transgenic technology has not yet been applied to the genomes of the cytoplasmic organelles (i.e., plastids and mitochondria) of higher plants.
The size of plastid DNA (ptDNA) in higher plants is in the range of 120 kb to 160 kb (Palmer, Ann. Rev. Genet., 19: 325-54, 1985), and encodes the genes involved in plastid maintenance and photosynthesis. By now, three plastid genomes have been sequenced, including that of Nicotania tabacum (Shinozaki et al., EMBO J., 5: 2043-49, 1986). In addition to photosynthesis, plastids serve as a compartment for amino acid and lipid biosynthesis. Most if not all the genes involved in these functions are encoded by the nucleus. The enzymes encoded by nuclear genes are synthesized on cytoplasmic ribosomes, and are subsequently transported into the plastids. Expression and accumulation of nuclear gene products and of plastid gene products is coordinated (Gruissem, Cell, 56: 161-70, 1989).
Formation of stably transformed plastid genomes requires integration of the transforming DNA by recombination. That recombination is a mechanism contributing to the evolution of plastid genome is evident when comparing genomes of different plant species. Also, continued recombination through the plastid inverted repeat has been described (Palmer, 1985, supra). It has also been shown that intergenomic plastid recombination occurs in heteroplastidic cells obtained by protoplast fusion (Thanh and Medgyesy, Plant Mol. Biol. 12: 87-93, 1989), and is extensive (Fejes et al., Theor. App. Genet. 79: 28-32, 1990).
Introduction and stable integration of exogenous DNA has been reported recently in the plastid genome of a unicellular alga, Chlamydomonas reinhardtii (Boynton et al., Science 240: 1534-38, 1988; Blowers et al., Plant Cell 1: 123-32, 1989). Initial success in transforming the plastid genome of Chlamydomonas by Boynton et al. was made possible by the development of a microprojectile DNA delivery system based on a particle gun that delivers DNA-coated tungsten microprojectiles into the cell. In addition, a powerful selection scheme was applied that relied on complementing nonphotosynthetic deletion mutants. Subsequently, Blowers et al. showed that the Chlamydomonas plastid genome can be expanded by integrating the coding sequence of an E. coli enzyme, neomycin phosphotransferase. Transformation of the psbA gene encoding the D1 reaction center polypeptide of photosystem II, and of the 16s rRNA gene conferring resistance to streptomycin and spectinomycin was reported by Boynton et al., p.p. 509-16 in Current Research in Photosynthesis, M. Baltsheffsky, ed., 1990.
In higher plant chloroplasts, only transient expression of introduced DNA has been reported. DNA uptake and transient expression by isolated cucumber etioplasts of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans, or of the E. coli enzyme, chloramphenicol acetyltransferase (CAT), has been reported (Daniell and McFadden, Proc. Natl. Acad. Sci. USA 84: 6349-53, 1987). The 5xe2x80x2 end of the psbA (pea), and rbcL (maize) plastid genes were fused with the CAT gene. Transient expression of the constructs in chloroplasts of cultured tobacco cells has been reported by Daniell et al., Proc. Natl. Acad. Sci. USA 87: 88-92 (1990) after biolistic delivery. Some of the vectors contained replication origins from ptDNA. CAT activity was sustained longer when the replicon origins were present. CAT activity, however, was not shown to be localized in chloroplasts. CAT activity, therefore, could have been the result of expression in the nucleus, since plastid gene promoters are known to support transcription initiation in the nucleus (Cornelissen and Vandewiele, Nucleic Acids Res. 17: 19-28, 1989).
European Application No. 87305573.5, filed Jun. 23, 1987, by M. C. Cannon and F. C. Cannon, describes a method for producing a plant whose cells express a desired gene by inserting the desired gene into the plastid genome of a plant cell. However, this application does not suggest a method for stably transforming plastids using nonlethal selection, nor does it provide any evidence suggesting that stable transformation was, or could be, achieved.
Transformation of plastids in higher plants was claimed after Agrobacterium-mediated transformation of N. tabacum (DeBlock et al., EMBO J. 4: 1367-72, 1985). A CAT gene was engineered for expression in the nucleus, and transgenic clones were selected for resistance to 10 xcexcg/ml chloramphenicol. The authors claim that there was fortuitous integration of the CAT gene into the ptDNA and expression from a plastid promoter. The line was reportedly unstable, and the authors"" claims have not been confirmed.
There are several differences between the Chlamydomonas system and higher plants that may be relevant for successful transformation of plastids. Two of these are discussed below. First, the number of plastids, and the number of plastid genomes per cell is much lower in Chlamydomonas than in Nicotiana. Chlamydomonas contains a single plastid, which carries up to 80 identical plastid genomes (Harris, The Chlamydomonas Sourcebook, p. 354, Academic Press, San Diego, 1989). In contrast, Nicotiana tabacum cells contain a variable number of plastids, about 100 in leaf cells, and 12 to 14 in meristematic cells and dedifferentiated tissue culture cells (Thomas and Rose, Planta 158: 329-38, 1983). In a study with cultured cells the number of plastid genome copies was estimated to be 3,000 to 12,000 per cell (Cannon et al., Plant Cell Reports 4: 41-45, 1985; Yasuda et al., Planta 174: 235-41, 1988).
Another important difference is that Chlamydomonas cells are grown photoautotrophically which allows stringent selection for photosynthetic ability, that is, functional plastids. This facilitates transformation by all proven methods, including Agrobacterium-mediated transformation (Weising et al., Ann. Rev. Genet. 22: 421-77, 1988), electroporation (Fromm et al., Nature 319: 791-93, 1986), calcium phosphate coprecipitation (Krens et al., Nature 296: 72-74, 1982), transformation by high-velocity microprojectiles (Klein et al., Proc. Natl. Acad. Sci. USA 85: 8502-05, 1988), and polyethylene glycol treatment (Negrutiu et al., Plant Mol. Biol. 8: 363-73, 1987). In contrast, higher plants are cultured photoheterotrophically, which reduces the stringency of selection for functional plastids.
Given the large number of plastid genomes in plant cells, the ability to select for the transformed genome in culture is a key element in achieving plastid transformation. Selection markers have been identified by screening culture plant cells for mutants resistant to various substances, such as antibiotics and herbicides. Since most of the selectable plastid genome markers have been developed through cell culture, it is not surprising that most are derived from Nicotania tabacum and Nicotiana plumbaginifolia, two species that are easy to grow in cell culture and to subsequently regenerate into plants. Resistance to inhibitors of plastid protein synthesis, conferred by mutation in the plastid 16S rRNA and 23S rRNA genes, are the most readily available markers. Other markers include resistance to streptomycin (Maliga et al., Nature 255: 401-02, 1973; Etzold et al., FEBS Lett. 219: 343-46, 1987; Fromm et al., Plant Mol. Biol. 12: 499-505, 1989), spectinomycin (Fromm et al., EMBO J. 6: 3233-37, 1987) and lincomycin (Cseplo and Maliga, Mol. Gen. Genet. 196: 407-12, 1984; Cseplo et al., Mol. Gen. Genet. 214: 295-99, 1988) which are the equivalent rRNA gene mutation used for transformation in Chlamydomonas (Harris et al., 1989, supra).
Plastid genome mutants resistant to triazine herbicides, have also been obtained in cultured Nicotiana cells. Triazine herbicides inhibit photosynthesis by interruption of electron flow at the acceptor of photosystem II. Selection was made feasible in Nicotiana cultures by lowering the concentration of sucrose in the medium, thereby making cellular proliferation partially dependent on photosynthesis (photomyxotrophic cultures; Cseplo et al., Mol. Gen. Genet. 200: 508-10, 1985; Sato et al., Mol. Gen. Genet. 214: 358-60, 1988). Selection for resistance to this class of herbicides is also a nonlethal color selection, resistant mutants being identified by their green color (Cseplo et al., 1985, supra). A mutation in two of the lines was localized to the psbA gene (Pay et al., Nucleic Acids Res. 16: 8176, 1988; Sato et al., 1988, supra). Similar mutant have been found in higher plants under field conditions (Maliga et al., p.p. 133-143 in Perspectives in Genetic and Biochemical Regulation of Photosynthesis, I. Zeitlich, ed., Alan R. Liss, N.Y., 1990), and isolated in Chlamydomonas (Erickson et al., Science 228: 204-07, 1985).
Naturally occurring resistance to tentoxin is also encoded on the plastic genome (Durbin and Uchytil, Biochem. Genet. 15: 1143-45, 1977). Pigment deficiency caused by plastome mutation is frequent, but does not appear to be a useful marker in culture. Pigment mutation in combination with antibiotic resistance mutations, however, have proved important in recovering a recombinant plastid genome (Medgyesy et al., Proc. Natl. Acad. Sci. USA 82: 6960-64, 1985).
The present invention provides a method for stable transformation of the plastids of higher plants. Others have attempted to obtain stable plastic transformation in higher plants, but without success. The present invention also provides efficient and versatile DNA constructs to accomplish stable transformation of plastid genomes and expression of foreign proteins in transformed plastids. These methods and constructs, heretofore unavailable, will enable improvement of useful plant species by genetic engineering of the plastid genome.
This invention provides DNA constructs and methods for stably transforming plastids of multicellular plants. The DNA constructs of the invention further enable high frequency stable plastid transformation and expression of foreign genes in plastids.
According to one aspect of the present invention, a DNA construct for stably transforming plastids of multicellular plants is provided. The DNA construct contains a transforming DNA, which comprises a targeting segment, a selectable marker gene and at least one cloning site adapted for insertion of an additional DNA segment. The targeting segment comprises a DNA sequence substantially homologous to a pre-determined plastid genomic sequence of a genome within a plastid to be transformed. The targeting segment is of sufficient size to promote homologous recombination with the pre-determined plastid genomic sequence, thereby replacing that sequence in the genome of the transformed plastid. The selectable marker gene is disposed within the targeting segment and confers a non-lethal selectable phenotype to cells containing plastids transformed with the DNA construct. The cloning sites for insertion of additional DNA segments are disposed within the targeting segment relative to the selectable marker gene so as not to interfere with the gene""s ability to confer the non-lethal selectable phenotype to the cells containing the transformed plastid.
According to another aspect of the present invention, a DNA construct for high frequency stable transformation of plastids of multicellular plants is provided. This DNA construct includes a targeting segment comprising a DNA sequence substantially homologous to a pre-determined plastid genomic sequence, as described above, of sufficient size to promote homologous recombination with the pre-determined plastid genomic sequence, thereby replacing that sequence in the transformed plastid genome. The transforming DNA further comprises a chimeric selectable marker gene disposed within the targeting segment at a position relative to each terminus of the targeting segment so as not to disrupt the homologous recombination. The chimeric selectable marker gene comprises a selectable marker coding segment encoding a gene product that confers a non-lethal selectable phenotype to cells containing plastids transformed with the DNA construct. The chimeric gene further comprises a 5xe2x80x2 regulatory segment, positioned relative to the selectable marker coding segment in the 5xe2x80x2 direction to promote expression of the selectable marker coding segment in the plastid. The chimeric gene also comprises a 3xe2x80x2 regulatory segment, positioned relative to the selectable marker coding segment in the 3xe2x80x2 direction to promote stability of mRNA produced during the expression of the selectable marker coding segment in the plastid. The target segment further comprises at least one cloning site for insertion of additional DNA segments, the cloning sites being disposed within the targeting segment relative to the chimeric selectable marker gene so as not to interfere with the ability of that gene to confer the non-lethal selectable phenotype to cells containing the transformed plastids.
In a preferred embodiment, the additional DNA segments added to the target segment are also chimeric genes, comprising (1) a coding segment encoding a gene product; (2) a 5xe2x80x2 regulatory segment positioned relative to the coding segment in the 5xe2x80x2 direction to promote expression of the coding segment in plastids; and (3) a 3xe2x80x2 regulatory segment positioned relative to the coding segment in the 3xe2x80x2 direction to promote stability of mRNA produced during expression of the coding segment in plastids.
According to another aspect of the present invention, a DNA construct is provided for stably transforming plastids of multicellular plants and expressing a gene product within the transformed plastids. This DNA construct comprises a transforming DNA having a targeting segment as described above. The transforming DNA further comprises a selectable marker gene disposed within the targeting segment, which confers a non-lethal selectable phenotype to cells containing plastids transformed with the DNA construct. The construct also contains at least one expressible DNA segment disposed within the targeting segment at a position that does not disrupt homologous recombination with the pre-determined plastid genomic region. The expressible DNA is also positioned relative to the selectable marker gene so as not to interfere with its ability to confer the non-lethal selectable phenotype to cells containing transformed plastids. The expressible DNA segment comprises (1) a coding segment encoding a gene product; (2) a 5xe2x80x2 regulatory segment positioned relative to the coding segment in the 5xe2x80x2 direction to promote expression of the coding segment in plastids; and (3) a 3xe2x80x2 regulatory segment, positioned relative to the coding segment in the 3xe2x80x2 direction to promote stability of mRNA produced during expression of the coding segment in the plastids.
In a preferred embodiment of the present invention, the DNA constructs described above are disposed within autonomously replicating vectors. These vectors are preferably of a type that facilitate manipulation of DNA segments contained therein by recombinant DNA techniques.
According to another aspect of the present invention, an expression cassette for producing a chimeric gene for expression in stably transformed plastids having genomes containing that gene is provided. The expression cassette comprises (1) a 5xe2x80x2 regulatory segment for controlling expression of a coding segment in plastids; (2) a 3xe2x80x2 regulatory segment for promoting stability of mRNA produced during expression of the coding segment in plastids; and (3) a cloning segment connecting the 5xe2x80x2 regulatory segment and the 3xe2x80x2 regulatory segment, and being adapted for insertion of a coding segment, such that expression of the inserted coding segment is controlled by the 5xe2x80x2 regulatory segment and the mRNA produced during that expression is stabilized by the 3xe2x80x2 regulatory segment.
In a preferred embodiment of the invention, the cloning segment of the aforementioned expression cassette further includes a stuffer segment. The stuffer segment is excised and replaced by a coding segment, to produce a chimeric gene for expression in plastids.
According to another aspect of the present invention, a 5xe2x80x2 regulatory segment for expressing a coding segment in plastids is provided. The 5xe2x80x2 regulatory segment controls expression of a coding segment in plastids when it is positioned relative to the coding segment in the 5xe2x80x2 direction to promote such expression. The 5xe2x80x2 regulatory segment comprises a promoter region and a 5xe2x80x2 untranslated region, with the promoter region being positioned immediately adjacent to the 5xe2x80x2 untranslated region in the 5xe2x80x2 direction. The 5xe2x80x2 untranslated region comprises a DNA sequence that encodes a ribosome binding site, and may also comprise other regulatory sequences. The 5xe2x80x2 untranslated region is also referred to herein as a xe2x80x9cleader sequence.xe2x80x9d
In a preferred embodiment, the 5xe2x80x2 regulatory segment described above further comprises a 5xe2x80x2 translated region positioned immediately adjacent to the 5xe2x80x2 untranslated region in the 3xe2x80x2 direction. This translated region comprises a translational start codon positioned in translational reading frame with the coding segment whose expression is controlled by the 5xe2x80x2 regulatory segment.
According to another aspect of the present invention, multicellular plant cells and plants having stably-transformed plastids may be obtained through the use of a DNA construct comprising a transforming DNA having a targeting segment, a selectable marker gene disposed within the targeting segment and at least one cloning site adapted for insertion of additional DNA segments, as described above. The transforming DNA is delivered into the plastid by one of several means known in the art, thereby enabling integration of the transforming DNA without interfering with the normal function of the plastid genome. Cells or tissues containing the potentially transformed plastids are placed on a non-lethal selection medium in which transformed plastids having the non-lethal selectable phenotype are preferentially maintained, while non-transformed plastids are lost. Cells or tissues are maintained on the selection medium until they have reached a homoplasmic condition, in which substantially all of the plastids of the cell or tissue have been transformed. The cells or tissues expressing the non-lethal selectable phenotype are selected, and thereafter may be regenerated to obtain plants.